The antigen-specific cytotoxicity mediated by cytolytic T lymphocytes (CTL) is an important cellular mechanism of antiviral and antitumor immune responses. The ultimate goal of our studies is to reveal the cellular and molecular requirements for CTL effector functions and differentiation of immature CD4+8+ thymocytes into functional CD8+ CTL. The exquisite specificity toward the targeted antigen is one of the most important properties of cell-mediated immune responses. However, we noticed that even highly specific CTL, which spare antigen-free target cells in the absence of antigen-bearing cells, could destroy the same antigen-free cells when they became "innocent" bystanders. We hypothesized that "innocent" bystander killing by activated CTL has to be tightly controlled and may play an important and yet to be clarified role in the mechanisms of immunopathologies. Fas-mediated signaling and perforin-mediated lysis of target cells account for most of the described cytotoxic consequences of CTL-target interactions and we investigated which of these cytotoxic mechanisms is responsible for the killing of innocent bystanders. No lysis of innocent bystanders was observed using i) functionally Fas-negative target cells (TC) from lpr mice; ii) anti-Fas mAb resistant lymphoma cells; iii) functionally FasL-deficient CTL from Gld mice. Normal CTL and CTL with disrupted perforin genes were both efficient in killing of innocent bystanders, leading to the conclusion that lysis of innocent bystanders is completely mediated by Fas, but not by perforin. Bystanders killing by CTL is tightly regulated by intracellular biochemical pathways, with the CTL's PP2a phosphatase playing the major role of intracellular "enforcer" of antigen-specificity during and/or after specific antigen-bearing target cell lysis. Our results are consistent with a model in which the innocent bystander killing of Fas-expressing cells by the CTL's surface Fas ligand proceeds until activated CTL are mutually eliminated by a Fas-mediated mechanism. The innocent bystanders are also partially protected from "dangerous" CTL by intracellular biochemical pathways that involve activities of the CTL's PP2a phosphatase. The future studies will focus on the understanding of molecular and cellular mechanisms and on the evaluation of immunopathological consequences of innocent bystanders' killing in vivo.